Chromatography material and a process of manufacturing that material

ABSTRACT

A process of manufacturing of a chromatography material comprising the steps of 
     (i) reacting a polymerisable at least bifunctional monomer A with a ligand also having a functional group which binds covalently with one of the functional groups of said polymerisable bifunctional monomer A,  
     (ii) to form a compound B comprising at least one polymerisable functional moiety  
     (iii) polymerizing the compound B essentially alone or with the polymerisable monomers in presence of porogenes to obtain a block of porous polymerisate which is self-supporting or  
     (iv) reacting the ligand and a spacer via a covalent bond which is cleavable to form a ligand-spacer conjugate and binding the ligand-spacer conjugate to the surface of a chromatographic support or reacting the ligand-spacer conjugate via a covalent bond to the at least bi-functional monomer A and polymerizing it essentially alone or with the polymerisable monomers in presence of porogenes to obtain a block of porous polymerisate which is self-supporting.

[0001] The invention concerns a process of manufacturing of achromatography material, a material obtainable according to the processan article comprising the material as well as a use of the material.

[0002] Chromatographic methods have been employed with great successwhenever mixtures of substances have to be separated. Typically, thechromatographic processes are performed on a solid matrix. The qualityof the separation and the respective reliability depends on the optimumchromatographic material. Mostly, the chromatographic matrices areporous matrices. By choosing the grade of porosity and/or the chemicalnature of the surface of the matrices the chromatographic processes canbe influenced and tailored for the respective separation problem. Forexample, if affinity chromatography is performed, an affinity ligand hasmostly to be immobilized at the surface of the chromatographic material.Also in catalysis performed on solid matrix the porosity and generalproperty of the surface of the matrix is important.

[0003] In these cases, for affinity chromatographic purposes as well asfor solid phase catalysis, the ligand as to be immobilized on therespective surface of the matrix. Typically, as solid matrices grainedmaterials having a certain particle size are used. The bond between theligand, for example the affinity ligand, and the solid matrix has to beat least as stable as that the material survives the separationconditions. Furthermore, if the immobilization of a ligand has to beperformed chemically, the reaction conditions have to be chosen in sucha way, that the binding properties of the ligand are not adverselyaffected.

[0004] For example, immobilization of polypeptides on chromatographicmedia happens in an undirected manner (Turková, 1978). Theimmobilization can only imperfectly be influenced by adjusting thereaction conditions. In the case of immobilization of polypeptides, itcan mostly only until a certain degree be determined which amino acidsof the proteins can be linked to the matrix via activated moieties. Itoften happens that the amino acids being responsible for the affinityinteraction between the ligand immobilized on the surface of the matrixand the molecule to be separated are not available anymore for specificreactions. Another difficulty may arise when the active part of theligand cannot be reached by the binding partner in the mixture to beseparated. Furthermore, due to the multipoint-attachment, thethree-dimensional structure of the ligand bound to the matrix may bealtered in such a way that the binding pocket will be deformed so thatthe substance to be separated cannot bind anymore (Walters, 1985).

[0005] In general the chromatographic media are designated in “gels” nomatter whether or not they are solid or soft gels or are built frommaterials which are completely different to the properties of a gel (forexample controlled pore glass). In solid gels such as Sepharose FastFlow (Pharmacia Biotech, Uppsala, Sweden) trisacryl (BioSepra Inc.Marlborough, USA) or Macro-prep (BioRad, Richmond, USA) the gel formingmacromolecules are arranged in bundles and the arrangement interactswith the fluidum of the mixture to be separated via capillary forces. Inthe soft gels (the genuine gels) like dextrane or polyacrylamid thepolymer interacts with the fluidum directly such as a liquid. The gelcan be interpreted as forming a single phase like a dynamic liquid(Jungbauer, 1994).

[0006] Typically, the following media are used in affinitychromatography: genuine gels from dextrane, agarose and polyacrylamid;silica material and chromatographic carriers which are stabilized via aceramic skeleton as well as so called perfusion gels which consists fromcoated polystyrene (Afeyan, 1990). An important criterion for achromatographic carrier or a carrier which can be used in the solidphase catalysis is its ability to be regenerated and, of course, anunspecific absorption as small as possible. Both criteria are determinedby the chemical nature of the support but also by the method of theimmobilization of the ligand on the surface of the carrier material aswell as the ligand itself (review for regeneration of differentchromatographic supports, Jungbauer, 1994). The method of regenerationof the matrix has to consider the chemical nature of the ligand and themethod how the ligand was bound to the matrix.

[0007] Basically, there can be differentiated two methods forimmobilization of ligands, one for inorganic carrier materials and amethod for immobilization of ligands on organic carriers. In the priorart, the immobilization of a ligand has been performed during amultistep procedure. In particular:

[0008] 1. introduction of an activatable moiety in or on the matrix, ifnecessary. The activatable moiety is normally a hydroxyl or a carboxylgroup. In most cases, the introduction of an activatable group isnecessary with inorganic substrates or supports. Also an activatedmoiety can be introduced. When bifunctional reagents are used for theactivation, the activation of the matrix and the introduction of anactivated “spacer” is performed in a one-step-reaction.

[0009] 2. the activation of the carrier is the first step with the mostorganic chromatographic substrates if no spacer is introduced with abifunctional reagent.

[0010] 3. introduction of a spacer having a reactive or activatablegroup. When the spacer does not have a reactive group, a second step foractivation has to be performed. If bifunctional reagents are used, step2 and step 3 are reduced to a single step.

[0011] 4. coupling of the ligand and

[0012] 5. blocking of the remaining reactive groups.

[0013] If a spacer is used which has two reactive groups, an activationhas not to be performed. In the case of organic substrate materials, thefirst step can be left out since free hydroxyl groups or otheractivatable groups are present.

[0014] Unfortunately, during coupling of a ligand on the matrix thishappens typically in such a way that it leads to an inhomogeneousdistribution of the ligand on the surface of the matrix (chromatographicsubstrate). For example the ligand concentration decreases from theouter to the inner portions of the particulate material. In this casethe ligates (the substances interacting with the ligands) are in contactwith a surface covered with the ligand with a density that is too highfor efficient binding. On the other hand in the inner portions of theparticular material the ligand density is too low. If block-polymers areutilized the problem may arise that during the immobilization process agradient of concentration of the ligand is established. Due to thisgradient, one will find an inhomogeneous distribution. Ligands for aprotein or a biopolymer are very often not available since they areimmobilized at sites which are not accessible for the proteins and otherbiopolymers.

[0015] It is very tedious to manufacture monoliths for affinitychromatography in high quantities with a reasonable batch to batchreproducibility. Each of the preactivated monoliths must be connected toa system equipped with at least one pump to deliver the reactionsolution, such as ligand mixture, each piece of monoliths must be testedindividually for its functionality such as dynamic binding capacity,ligand density, leakage and/or resolution.

[0016] Therefore, it is the object of the invention to provide a processof manufacturing of a chromatography material avoiding the abovementioned drawbacks and to improve the ligand density which is necessaryfor an optimal separation of mixtures or conversion of substrates, andto provide a material which can be used as a chromatographic carrier orsubstrate for conversion of substances.

[0017] According to the invention this object is achieved by a processof manufacturing of a chromatography material comprising the steps of

[0018] (i) reacting a polymerisable at least bifunctional monomer A witha ligand also having a functional group which binds covalently with oneof the functional groups of said polymerisable bifunctional monomer A,

[0019] (ii) to a compound B comprising at least one polymerisablefunctional moiety

[0020] (iii) polymerizing the compound B essentially alone or with thepolymerisable monomers in presence of porogenes to obtain a block ofporous polymerisate which is self-supporting or

[0021] (iv) reacting the ligand and a spacer via a covalent bond whichis cleavable to form a ligand-spacer conjugate and binding theligand-spacer conjugate to the surface of a chromatographic support orreacting the ligand-spacer conjugate via a covalent bond to the at leastbi-functional monomer A and polymerizing it essentially alone or withthe polymerisable monomers in presence of porogenes to obtain a block ofporous polymerisate which is self-supporting.

[0022] According to the invention, a ligand is bound to a reactivemonomer. In order to improve the accessibility of the ligand it islinked to a spacer forming a ligand spacer conjugate. This spacer can becleaved after polymerization reaction and be removed. After forming ofthe polymerisate to the respective material such as particulate materialor a monolithic block, the polymerisate is ready for use as separationmedium, for example in affinity chromatography, in the reactivechromatography or as catalyst.

[0023] The spacer can also be bound directly to the ligand. In this casethe respective derivative is bound to a conventional matrix. In thiscase, only at such sites ligands are immobilized which are accessiblefor large molecules such as proteins or other biopolymers since theligand can only reach pores of appropriate dimension. The extension ofthe spacer is selected in a way depending of the extension of thebiomolecule or protein to be bound on the chromatographic matrix. Alsothe pore sizes and the pore structures of the separation medium has tobe considered.

[0024]FIG. 1 shows a typical process of the invention for themanufacturing of a chromatographic material or catalyst according to thefirst alternative of the main claim. A ligand having an amino group of asulfidyl group (according to FIG. 1 H₂N−peptide−COO⁻) is reacted withglycidyl methacrylate and the resulting conjugate is further processafter separation of byproducts. A macromolecule which does not effectthe polymerization reaction (leading to a blockpolymer) negatively isfunctionalized with a functional group. This functional group effectsthe reversible linkage of the macromolecule to the conjugate. Accordingto FIG. 1, the macromolecule not interfering with the polymerizationreaction is polyethylene glycol. Typically, the reaction product ispurified and polymerized to a block. The macromolecule is cleaved offand removed.

[0025] The resulting material block of polymerisate can be used foraffinity chromatography, reactive chromatography or catalyst.

[0026] Alternatively, the ligand can be coupled to a macromolecule. Thisresults in an increase of the molecular radius and immobilization of themacromolecule-ligand conjugate happens only at those sites which areaccessible for a protein. This situation is shown in FIG. 2. A peptideligand having an increased size due to linkage to a spacer wasimmobilized at the surface of a conventional matrix.

[0027] The invention provides the advantage that the ligand is onlyimmobilized at those sites which are accessible for the ligate. Due tothis the capacity is increased, difficult ligands can be utilized, thein most cases expensive ligand is saved as well as non-specificadsorption is reduced since less amount of ligand is employed.

[0028] A monomer D may be present in steps (iii) or (iv) which monomeris crosslinkable. The monomers modify the properties of thepolymerisate.

[0029] In a preferred embodiment of the process of the invention atleast one further monomer C is present.

[0030] Preferably, the ligand is an affinity ligand. Typically, theaffinity ligand comprises biospecificity, immunoaffinity,enzyme-substrate affinity, receptor-ligand affinity or nucleotideaffinity, such as hybridisation, as well as specific ionic interactionssuch as ion exchange interactions.

[0031] In particular, the bifunctional monomer A is glycidylmethacrylate, styrene ring substituted styrenes wherein the substitutioninclude but is not limited to Chloromethyl, alkyl with up to 18 carbonatoms, hydroxyl, t-butyloxicarbonyl, halogen, nitro, amino group,protected hydroxyls or amino groups, vinylnaphthalene, acrylates,methacrylates, vinylacetate and pyrrolidone, and combinations thereof.

[0032] The crosslinkable co-monomer D is preferably ethylene glycoldimethacrylate, divinylbenzene, divinylnaphtalene, divinylpyridine,alkylene dimethacrylates, hydroxyalkylene dimethacrylates,hydroxyalkylene diacrylates, oligoethylene glycol diacrylates, vinylpolycarboxylic acids, divinyl ether, pentaerythritol di-,tri-, or tetramethacrylate or acrylate, trimethylopropane trimethacrylate or acrylate,alkylene bis acrylamides or methacrylamides, and mixtures of any suchsuitable polyvinyl monomers.

[0033] In a preferred embodiment of the process of the invention theligand or the compound B is bound with a spacer via a covalent bondwhich is cleavable under reaction conditions not employed duringpolymerization reaction of the compound B.

[0034] The spacer is selected considering the pore size of thechromatographic material to be manufactured.

[0035] According to the invention a preferred spacer is a polyethyleneglycol optionally functionalized with a group cleaveable by a dilutehalogenated organic acid such as trifluor acetic acid (TFA). Ascleaveable groups preferably 4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB), 3-(4-Hydroxymethylphenoxy) propionic acid (PAB),3-Methoxy-4-hydroxymethylphenoxy acetic acid,4-(2′,4′-Dimethoxyphenylhydroxylmethyl) phenoxymethyl or2-Methoxy-4-alkoxybenzyl alcohol are used.

[0036] These cleaveable groups are activated through a hydroxy grouppresent in the cleaveable group by carbodiimides such asN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodimide(DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) orcarbonyldiimidazole.

[0037] To this reaction mixture compounds such as 1-hydroxybenzotriazole(HOBt), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),2-(H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(HBTU) 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), and2-[2-oxo-1(2H)-pyridyl]-1,1,3,3-bispenta-methyleneuroniumtetrafluoroborate (TOPPipU) accelerating carbodiimide-mediated couplingare added.

[0038] In order to improve the process of the invention it is possiblethat the reaction product of monomer A, ligand or compound B, optionallywith linked spacer is purified after reaction. It can also beadvantageous that the products of the polymerization reaction after step(iii) are washed with methanol and water.

[0039] In the process of the invention typically porogenes such asdodecanol, cyclohexanol, tetradecanol, toluol, isooctanol, hexanol,methanol, ethanol, propanol, butanol or isopropanol.

[0040] A material obtainable by the process of the invention is alsosubject of this invention. The material of the invention can be used forchromatography, performing conversion reactions which are employingactive surfaces strong acids to donate protons to a reactant and to takethem back, or bases to catalyse processes including isomerization andoligomerization of olefins, reaction of olefins with aromatics,hydrogenation of polynuclear aromatics, esterification andetherification, or sulfides for weak redox reaction.

[0041] Furthermore, the material of the invention can be employed forthe purification of plasma proteins, recombinant proteins, plantprotein, bacterial proteins, nucleic acids such as plasmids and cosmids,peptides peptoides, oligonucleotides, oligosaccharides, polysaccharides,fatty acids, steroids.

[0042] An article comprising the material of the invention in a housinghaving one inlet and one outlet for liquids applied is also subject ofthe invention.

[0043] With the material of the invention for instance a rod shapedmonolith is polymerised carrying the affinity ligand. Due to thereproducibility of the method of the invention it is sufficient to testrepresentative samples which are tested for functionality, such asdynamic binding capacity, ligand density, leakage and/or resolution. Therod can be tailored to a desired geometry according to the requirementsof the user. These pieces are mounted into a housing and are ready touse without further testing of each individual formed piece.

[0044] The invention is further explained by the following, non-limitingexamples.

EXAMPLE 1

[0045] Preparation of a Conjugate and a Blockpolymer.

[0046] The peptide having the amino acid sequence NH₂-YLSYPLTFGA(Welling, 1991) having an affinity to lysozyme was reacted with glycidylmethacrylate. The peptide was dissolved in 0.1 M sodium carbonatebuffer, pH 10.0 to which 0.15 M sodium chloride was added. Thereafter,glycidyl methacrylate was added in a 100 times excess. To this reactionmixture acetonitril was added to form a homogenous solution. Thereaction was performed at room temperature for at least 48 hours undervigorous stirring. Thereafter, the conjugate was purified by means ofpreparative reversed phase chromatography (RPC) and byproducts as wellas the oligomers were removed. The RPC was performed on Nucleosil102/5/C18, column 16 mm I.D.×250 mm. The column was conditioned with0.1% TFA/water and after sample loading eluted withinacetonitril/trifluoroacetic acid (TFA) gradient. The eluate wasmonitored with an UV-monitor at 214 nm. A typical chromatograph is shownin FIG. 3.

[0047]FIG. 3 shows the purification of a glycidyl methacrylate conjugateby means of reversed phase chromatography on a Nucleosil C18 column.Based on 50 mg peptide the conjugate was loaded oh the column and elutedwithin an acetonitril gradient. The fractions were collected andanalyzed for molecular weight by means of MALDI-TOF. The respective massspectra are shown in FIG. 4. FIG. 4a shows the peak I and 4 b the peakII of FIG. 3.

[0048] The peptide used has a theoretical relative molecular mass of1131, glycidyl methacrylate of 142.13, the conjugate consisting of 1molecule peptide and 1 molecule methacrylate giving a theoreticalrelative molecular mass of 1273. When the peptide is conjugated with twomolecules methacrylate the relative molecular mass is 1415. As can bederived from FIGS. 3 and 4, the conjugate of free peptide and conjugateof two methacrylate residues was separated. The pool with the singleconjugate shows a molecular mass of 1274 (H⁺). Peak II in FIG. 3contains a substance having a relative molecular mass of 1416 (H⁺) whichis corresponding to a conjugate having two glycidyl methacrylateresidues.

[0049] Fraction I is mixed with 100 ppm referred to the conjugate 2.6Di-tert-butyl-4-methylphenol. This is advisable in order to avoid aprepolymerisation during the subsequent lyophilization. After removal ofthe solvent, the peptide glycidyl methacrylate conjugate is ready forpolymerization.

[0050] The conjugate is polymerized in different ratios together withglycidyl methacrylate and ethylene glycol dimethacrylate at 6520 C.Cyclohexanol and Dodecanol are serving as so called porogens.Benzoylperoxide is used as polymerisation initiator. The resultingreaction product is a blockpolymer. The peptide-glycidyl methacrylateconjugate is used in different concentrations by substituting therespective glycidyl methacrylate portion. Up to 25 mg conjugate per mlpolymerisate were used. Thereafter, the polymer block was washed withmethanol and sliced in cylindrical disks. These disks were sealed withan O-ring, and put into HPLC-cartridges. The polymerisate was tested bymeans of affinity chromatography. For the determination of the bindingcapacity a break-through curve was utilized.

EXAMPLE 2

[0051] Determination of Functionality

[0052] In order to test the functionality of the blockpolymer accordingto example 1, a break-through curve was determined using lysozyme (FIG.5). This curve was compared with a curve of non-derivatised monoliths(blockpolymers). The monoliths were regenerated with a 1 M sodiumchloride solution and, thereafter, conditioned with a 20 mMtris/HCl-buffer, pH 7.5. The polymer was loaded with lysozyme dissolvedin the conditioning buffer. The concentration of lysozyme was 0.2 mg/ml,the flow rate 2 ml/min. The bed volume was 1.5 cm (330 μl). The bindingcapacity resulted in a value of 1.6 mg lysozyme referring to 1 mlpolymer. The capacity was calculated based on the value at 10% of themaximal absorption.

EXAMPLE 3

[0053] Peptide Ligand with Spacer.

[0054] The ligand or conjugate was convalently linked to a spacer. Theresulting compound was blockpolymerized. The conjugate of peptide andglycidyl methacrylate was synthesized according to example 1. Then thefree carboxyl group of the conjugate is activated withdicyclohexylcarbodiimide (DCC) and hydroxybenzotriazol (HBT) and linkedto a modified polyethylene glycol. The modified PEG is an amino PEGderivatised with 4-hydroxy-3-methoxyphenoxybuturic acid. The activatedcarboxyl group of the peptide glycidyl methacrylate conjugate iscovalently linked to the free hydroxyl group of the modifiedpolyethylene glycol. This covalent bond may be cleaved in presence of aweak acid. The peptide glycidyl methacrylate-polyethylene glycolconjugate was precipitated by addition of diethylether and, thereafter,purified by means of size exclusion chromatography with a superdex 30(Amersham-Pharmacia Biotech) column. Byproducts and excess reagents wereremoved. In addition, at first step FMOC-β-Alanin can be introduced inorder to increase the yield of the coupling reaction. After cleavage ofthe FMOC moiety by means of 20% piperidin a terminal amino group isgenerated. The yield of the coupling reaction using an amino groupinstead of an OH-group is larger. After lyophilization which again isperformed in the presence of 100 ppm 2,6 Di-tert-butyl-4-methylphenol,the conjugate (consisting of methacrylate residue, a peptide andpolyethylene glycol as spacer) is yielding a blockpolymer.

[0055] Unreacted products and byproducts were removed and thepolyethylene glycol was cleaved by reacting with 1% TFA. The cleaved PEGwas washed away with methanol and 1 M sodium chloride solution. Thefunctionality of the affinity substrate was determined by means of anaffinity chromatography experiment.

EXAMPLE 4

[0056] Determination of Functionality with a Break-through Curve

[0057] The functionality of the blockpolymer with spacer according toexample was tested by frontal chromatography and breakthrough oflysozyme was used as a measure for binding capacity (FIG. 6). Thebreakthrough was monitored at 280 nm using a on-line UV-monitor (UV-1,Amersham-Pharmacia Biotech, Uppsala, Sweden). This breakthrough curvewas compared with a curve of non-derivatised monoliths (blockpolymers).The monoliths were regenerated with a 1 M sodium chloride solutioncontaining a 20 mM Tris/HCl-buffer, pH 7.5 conditioned with a 20 mMTris/HCl-buffer, pH 7.5. The polymer was loaded with lysozyme dissolvedin the conditioning buffer. The concentration of lysozyme was 0.2 mg/ml,the flow rate 2 ml/min. The bed volume was 1.5 cm (330 μl). The bindingcapacity resulted in a value of 1.0 mg lysozyme referring to 1 mlpolymer. The capacity was calculated based on the value at 10% of themaximal absorption.

EXAMPLE 5

[0058] Determination of Pore Size Distribution.

[0059] Pore size distribution of the material described in the examples1 to 5 was determined with mercury porosimetry. The experiments wereperformed on mercury porosimetry PASCAL 440 (Porotech). The driedpolymer is suspended in liquid mercury. The volume of the intrudedmercury is a direct measure for the penetrated pore volume. In FIG. 6,the pore size distribution of monoliths according to the invention isshown. As reference a CIM-expoxy material from BIA Separations,Ljubljana (Slovenia) was used. The pore size distribution of thematerial made with the conjugate is significantly higher and thereforeeasier accessible than the conventional material used as a reference.

EXAMPLE 6

[0060] Immobilization of a Peptide Synthetic Polymer Conjugate on aConventional Substrate.

[0061] The peptide of example 1 is dissolved in a sodium chloridebuffer, pH 5.0 having 20% acetonitril. The N-terminal of the peptide isblocked with fluorenyl-methyl-oxycarbonyl-N-hydroxy-succinimide. Thenthe N-terminal blocked peptide is purified by removing excessfluorenyl-methyl-oxycarbonyl-N-hydroxy-succinimide by means of reversedphase chromatography followed by activation of the C-terminus withdicyclohexylcarbodiimide and HBT under addition of triethylamine for atime period of 2 hours. The activated peptide is reacted at roomtemperature and stirring with the derivatized polyethylene glycol. After20 hours, the reaction is finalized, the FMOC group cleaved by piperidinand the peptide-polyethylene-glycol-conjugate is precipitated withdiethyl ether. To 1 ml reaction solution 3 ml diethyl ether is added andfurther purified with size exclusion chromatography. The purifiedconjugate having a free N-terminus is immobilized on an epoxy activatedmatrix at pH 10 in aqueous sodium chloride buffer during a time periodof 48 hours. The PEG is cleaved by addition of 1% TFA for 1 hour.Thereafter, the affinity column is thoroughly washed with aqueous sodiumchloride buffer.

EXAMPLE 7

[0062] Determination of Functionality with a Breakthrough Curve

[0063] The gel with the immobilized peptide of example 6 was packed intoa HR 5 column (Amersham Pharmacia Biotech, Uppsala, Sweden). The gel wasthen equilibrated with a 20 mM Tris/HCL-buffer, pH 7.5 and then loadedwith lysozyme dissolved in the equilibration buffer. The concentrationwas 0.1 mg/ml and the flow rate 0.33 ml/min. The binding capacity wascalculated with 5.5 mg/ml gel. The capacity was compared with a gelwhere 20 mg of the peptide were immobilized in a conventional way on thesame type of material. In this case the capacity was only 2.7 mg/ml gel(FIG. 7).

EXAMPLE 8

[0064] Site-Directed Immobilization of a Peptide Synthetic PolymerConjugate with Protecting Groups on a Conventional Substrate

[0065] The peptide with the sequence H₂N-EYKSWEYC-COOH, which bindscoagulation F VIII (Amatscheck et al.) is synthesized with Fmocchemistry. The lysine side chain is protected with4,4-(dimethyl-2,6-dioxocyclohexylidene)-ethyl protecting group (Dde).Cystein is protected with the t-Butylthio protecting group. The fullprotected peptide is cleaved off the resin with 1% TFA. The Ddeprotecting group is removed by treatment with hydrazin. The reactionmixture is purified by a gel filtration step and the purified peptide isactivated with dicyclohesylcarbodiimide and HOBt according to example 6.The peptide is then reacted at room temperature and stirring with thederivatized polyethylenglycol. After 20 hours thepeptide-polyethylenglycol-conjugate is precipitated with diethyl ether,dissolved in water and purified with a gel filtration step. The purifiedconjugate, with the free amino group of the lysine side chain is thenreacted with an epoxy activated matrix. After 48 hours of reaction theremaining protecting groups are removed by treatment with 20% piperidinto remove the Fmoc group and then 100% TFA to remove other protectinggroups and to cleave off the polyethylenglycol from the immobilizedpeptide.

[0066] The functionality of the chromatography is tested with apre-purified F VIII preparate. F VIII binds specifically to the peptide,impurities can be detected in the unbound fraction (FIG. 8).

[0067] List of References

[0068] Afeyan, N. B., Gordon, N. F., Mazsaroff, I., Varady, L., Fulton,S. P., Yang, Y. B. and Regnier, F. E. (1990) Flow-though particles forthe high-performance liquid chromatographic separation of biomolecules:perfusion chromatography Journal of Chromatography, 519, 1-29.

[0069] Jungbauer, A. and Boschetti, E. (1994) Manufacture of recombinantproteins with safe and validated chromatographic sorbents Journal ofChromatography. B. Biomedical Applications, 662, 143-79.

[0070] Turkova, J. (1978) Affinity Chromatography. Elsevier, Amsterdam,N.Y.

[0071] Walters, R. R. (1985) Affinity chromatography AnalyticalChemistry, 57, 1099A-1101A.

[0072] Welling, G. W., van Gorkum, J., Damof, R. A., Drijfhout, J. W.,Bloemhoff, W. and Welling-Wester, S. (1991) A ten-residue fragment of anantibody (mini-antibody) directed against lysozyme as ligand inimmunoaffinity chromatography Journal of Chromatography, 548, 235-42.

1. A process of manufacturing of a chromatography material comprisingthe steps of (i) reacting a polymerisable at least bifunctional monomerA with a ligand also having a functional group which binds covalentlywith one of the functional groups of said polymerisable bifunctionalmonomer A, (ii) to form a compound B comprising at least onepolymerisable functional moiety (iii) polymerizing the compound Bessentially alone or with the polymerisable monomers in presence ofporogenes to obtain a block of porous polymerisate which isself-supporting or (iv) reacting the ligand and a spacer via a covalentbond which is cleavable to form a ligand-spacer conjugate and bindingthe ligand-spacer conjugate to the surface of a chromatographic supportor reacting the ligand-spacer conjugate via a covalent bond to the atleast bifunctional monomer A and polymerizing it essentially alone orwith the polymerisable monomers in presence of porogenes to obtain ablock of porous polymerisate which is self-supporting.
 2. The process ofclaim 1 wherein in steps (iii) or (iv) a monomer D is present which iscrosslinkable.
 3. The process of claim 1 additionally providing at leastone further monomer C.
 4. The process of claim 3 wherein the ligand isan affinity ligand.
 5. The process of claim 4 wherein the affinityligand comprises biospecificity, immunoaffinity, enzyme-substrateaffinity, receptor-ligand affinity or nucleotide affinity, such ashybridisation.
 6. The process of claim 3 wherein the affinity ligandcomprises specific ionic interactions such as ion exchange interactions.7. The process of claim 1 wherein the bifunctional monomer A is glycidylmethacrylate, styrene ring substituted styrenes wherein the substitutionincluding but not limited to Chloromethyl, alkyl with up to 18 carbonatoms, hydroxyl, t-butyloxicarbonyl, halogen, nitro, amino group,protected hydroxyls or amino groups, vinylnaphthalene, acrylates,methacrylates, vinylacetate and pyrrolidone, and combinations thereof.8. The process of claim 2 wherein the crosslinkable comonomer D isethylene glycol dimethacrylate, divinylbenzene, divinylnaphtalene,divinylpyridine, alkylene dimethacrylates, hydroxyalkylenedimethacrylates, hydroxyalkylene diacrylates, oligoethylene glycoldiacrylates, vinyl polycarboxylic acids, divinyl ether, pentaerythritoldi-, tri-, or tetra methacrylate or acrylate, trimethylopropanetrimethacrylate or acrylate, alkylene bis acrylamides ormethacrylamides, and combinations of any such suitable polyvinylmonomers.
 9. The process of claim 1, wherein the ligand or the compoundB is bound with a spacer via a covalent bond which is cleavable underreaction conditions not employed during polymerization reaction of thecompound B.
 10. The process of claim 1, wherein the spacer is selectedconsidering the pore size of the chromatographic material to bemanufactured.
 11. The process of claim 9 wherein the spacer is apolyethlene glycol optionally functionalized with a group cleaveable bya dilute halogenated organic acid such as trifluor acetic acid (TFA). Ascleaveable groups preferably 4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB), 3-(4-Hydroxymethylphenoxy) propionic acid (PAB),3-Methoxy-4-hydroxymethylphenoxy acetic acid,4-(2′,4′-Dimethoxyphenylhydroxyl-methyl) phenoxymethyl or2-Methoxy-4-alkoxybenzyl alcohol are used.
 12. The process of claim 1wherein the reaction product of monomer A with a ligand to form compoundB, optionally with linked spacer, is purified after reaction.
 13. Theprocess of claim 1 wherein the products of the polymerization reactionafter step (iii) are purified.
 14. The process of claim 1 whereinporogenes are selected from the group consisting of dodecanol,cyclohexanol, tetradecanol, toluol, isooctanol, hexanol, methanol,ethanol, propanol, butanol or isopropanol.
 15. A material obtainableaccording to one of the preceding claims.
 16. An article comprising thematerial of claim 15 in a housing having one inlet and one outlet forliquids to be applied.
 17. Use of a material according to claim 15 forchromatography and performing conversion reactions which are employingactive surfaces strong acids to donate protons to a reactant and to takethem back, or bases to catalyse processes including isomerization andoligomerization of olefins, reaction of olefins with aromatics,hydrogenation of polynuclear aromatics, esterification andetherification, or sulfides for weak redox reaction.
 18. Use of amaterial according to claim 15 for the purification of recombinantproteins, immunglobulins, antibodies, plasma proteins, in particularproteins involved in the blood clotting cascade such as clotting factorsand/or inhibitors.